Molecular two-photon absorption was predicted by Goppert-Mayer in 1931. Upon the invention of pulsed ruby lasers in 1960, experimental observation of two-photon absorption became a reality. Subsequently, two-photon excitation has found application in biology and optical data storage, as well as in other fields.
There are two key differences between two-photon-induced photoprocesses and single-photon induced processes. Whereas single-photon absorption scales linearly with the intensity of the incident light, two-photon absorption scales quadratically. Higher-order absorptions scale with a related higher power of incident intensity. As a result, it is possible to perform photoreactive processes with three-dimensional spatial resolution. Also, because photoreactive processes involve the simultaneous absorption of two or more photons, the absorbing chromophore is excited with a number of photons whose total energy equals the equals the energy of an excited state of a photoreactive photosensitizer, even though each photon individually has insufficient energy to excite the chromophore. Because the exciting light is not attenuated by single-photon absorption within a photoreactive matrix or material, it is possible to selectively excite molecules at a greater depth within a material than would be possible via single-photon excitation by use of a beam that is focused to that depth in the material. These two phenomena also apply, for example, to excitation within tissue or other biological materials.
Major benefits have been achieved by applying photoreactive absorption to the areas of photoreacting and microfabrication. For example, in photoreactive lithography or stereolithography, the nonlinear scaling of photoreactive absorption with intensity has provided the ability to write features having a size that is less than the diffraction limit of the light utilized, as well as the ability to write features in three dimensions (which is also of interest for holography). Such work has been limited, however, to slow writing speeds and high laser powers. At high laser powers, one-photon absorption may occur causing local heating and/or sample damage. Thus there is a need to enhance the peak intensity at the volume of interest while keeping the overall intensity low elsewhere.
Furthermore, the z-axis resolution in two photon microfabrication may be limited by the material but more usually is defined by the optics. In most of the published art, the depth of the reacted voxel (the smallest three dimensional volume element) is defined by the numerical aperture (NA) of the focusing optics and is proportional to the square of the NA. The lateral extent of the reacted voxel is directly proportional to the NA, so the depth and width of the reacted voxel cannot be varied independently (at a fixed wavelength). Creating a shallow depth of field necessitates a small lateral dimension to the voxel, and a small voxel means longer writing times for large structures.
S. Hell and E. H. K. Stelzer in “Fundamental Improvement of Resolution With a 4Pi-Confocal Fluorescence Microscope Using Two-Photon Excitation”, Optics Communications, 93, 277-282 (1992), describe using interference between two counter propagating beams, that is two beams with the propagating vectors at 180 degrees to each other, to enhance the z-axis resolution in two-photon fluorescence microscopy. In International Publication No. WO 99/54784 by Goodman and Campagnola, the use of two counter propagating beams is described with regards to enhanced z-axis resolution of a two photon microfabrication process. The described apparatus involves two high numerical aperture microscope objectives on opposite sides of a sample on a moveable stage. Such a configuration allows limited choice in the shape of the reacted region. Furthermore, it may not be used with non-transparent substrates.
Thus, there is a need for alternative methods of achieving spatial localization without sacrificing writing speed or flexibility in substrate choice. in exposing photoreactive absorption compositions.